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Title: A comparison of IL-1β and IL-8 mRNA and their respective
proteins in equine bronchoalveolar lavage fluid during recurrent airway
Authors: Sarli Giuseppe, Peli Angelo , Marrocco Romina , Zanotti Lucia , Ducci Alice1 , Isani Gloria , Alessandra Scagliarini , Sassi Francesco , Benazzi Cinzia , Cinotti Stefano , Pietra Marco S
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4.REPORT: This work is a study of non-cytokine proteins that could be involved in inflammation as well as their related mRna in stabled horses as distinct to cytokines (mRna). The findings could be relevant due to the importance of the conditions in stabled horses worldwide. The number of animals compared is not ideal and as noted in instructions for authors would have been better to have two populations of 8 each. However the statistical methods used were appropiate. The reviewer notes the difficulty in obtaining equines for this type of work and a minimum of 4 in this case is acceptable for control pasture group 2. In my view, at most, the results are only suggestive of a role for non-macrophage cells involvement inducing RAO. ABSTRACT. Some conclusions are required here see suggested changes below. INTRODUCTION. NA, MATERIALS and METHODS NA, RESULTS NA, DISCUSSION NA, REF NA accpt with minor modifications WR.
The role of different cytokines in triggering airway inflammation during recurrent airway obstruction (RAO) has become the focus of recent investigations. The majority of published papers are principally concerned with cytokine mRNA while there are fewer papers which deal with their respective proteins alone or their correlation with mRNA in the same trial. In the present study IL-1β and IL-8 mRNAs and their respective proteins were quantified by Real Time RT-PCR and western blot and related to bronchoalveolar lavage fluid (BALF) cytology in 8 horses during an RAO attack induced by stabling on straw. The results were compared to those obtained from 4 susceptible horses kept in a pasture. Significant changes in cytokine profile and BALF pattern were observed in the challenge group during the study (12 days) while no modifications were detected in the control group. An increase in IL-1β mRNA and protein preceded the increase of the same parameters as IL-8, but the related mRNA and respective protein levels did not strictly follow the same pattern. The rise in IL-1β protein, but not mRNA, continued over the entire period and was correlated with BALF neutrophilia. On the contrary, the increase in both IL-8 mRNA and protein were transient, and the peak of the protein (day 8) followed the peak of BALF neutrophilia.
Suggestion: Somehow try to include SOME of this information in ABSTRACT
The increase of IL-1β, paralleled by neutrophil and epithelial cell accumulation
in the BALF, is indicative of the active production of the cytokine by these
cells. The accumulation of neutrophils is recorded earlier than the increase of
IL-8 indicating the presence of different chemokines for neutrophils in the
early phase of the disease. Even if the amounts of pro-inflammatory cytokines
produced by cells other than macrophages, namely neutrophils and epithelial
cells, is low, their elevated number could result in a cumulative amount higher
than that of macrophages. This reinforces the pivotal role of cells other than
the macrophages in driving airway inflammation during RAO.
Recurrent Airway Obstruction (RAO) is a major equine affliction associated with stabling, and it is a common cause of exercise intolerance and poor performance in horses which frequently leads to eliminating animals from competitions [Sasse, 2001; Beech, 2002]. Although most Authors consider this disorder as a valid model of spontaneous disease for studying the asthma in humans, the exact aetiopathogenesis is still under debate and many factors have been suggested to have a role in its etiology [Ainsworth et al., 2006, 2007; Deaton et al., 2007; Jost et al., 2007].
The role of different cytokines in triggering airway inflammation during RAO has become the focus of recent investigation. The majority of published papers are principally concerned with cytokine mRNA [Franchini et al., 1998; Lavoie et al., 2001; Ainsworth et al., 2003; Cordeau et al., 2004; Horohov et al., 2005; Kleiber et al., 2005; Laan et al, 2006; Riihimäki et al., 2008] while there are fewer papers which deal with their respective proteins alone [Franchini et al., 2000, Desjardins et al., 2004] or their correlation with mRNA in the same trial [Ainsworth et al., 2006; Gigučre et al., 2002; Perkins et al., 2008].
The cytokines investigated are mainly IL-4, IL-2, IL-17 and IFN-γ [Ainsworth et al., 2006, 2007; Lavoie et al., 2001; Ainsworth et al., 2003; Cordeau et al., 2004; Horohov et al., 2005,; Kleiber et al., 2005] with the aim of demonstrating a Th1- or a Th2-dependent pathogenetic mechanism; however, a final conclusion has not been reached and various responses have been proposed [Horohov et al., 2005].
In several studies pro-inflammatory cytokines (IL-1β and IL-8) have been investigated by different ways: only as the mRNA [IL-1β, Laan et al., 2006; Gigučre et al., 2002; Pietra et al., 2007; IL-8, Franchini et al., 1998; Ainsworth et al., 2003; Laan et al, 2006; Pietra et al., 2007; Riihimäki et al., 2008] or protein [IL-8, Desjardins et al., 2004; Gigučre et al., 2002] and in a minor extent comparing mRNA and protein [IL-8, Ainsworth et al., 2006; Gigučre et al., 2002; Perkins et al., 2008]. Considering the post-transcriptional mechanisms operating in gene expression, it is necessary to design studies where both mRNAs and their respective proteins are investigated. This is due to the fact that evidence of gene expression does not prove active synthesis of the relevant protein. Considering that the majority of the studies on RAO and cytokines concern the transcript, they do not provide certain information on the role of the active cytokine. Furthermore, it is difficult to compare the data available in the literature because the times of sampling after RAO induction are very different ranging from 1 day to 7 weeks post challenge [Ainsworth et al., 2003, 2006; Kleiber et al., 2005; Laan et al, 2006; Desjardins et al., 2004; Gigučre et al., 2002].
Cytokines have a short half-life (hours) [Verfallie et al., 2001] and long and very different post-challenge times of sampling can give scant information on their possible role. In view of the limited information concerning mRNA-protein relationships of pro-inflammatory cytokines in RAO, the aim of the present paper was to evaluate mRNA and protein for IL-1β and IL-8 in the BALF of RAO-induced horses, and the comparison of the results with the BALF cell modification. The data came from an initial challenge of horses stabled on moldy hay which had an anamnesis of RAO; during the time of this challenge several BALF samplings were planned to investigate IL-1β mRNA and IL-8 mRNA and their respective proteins. A second set of data were obtained by immunohistochemical investigation on BALF cytospin slides of an RAO-affected horse to ascertain the cell-type containing IL-1β and IL-8 protein.
MATERIALS AND METHODS
Statement of animal care
All experimental procedures were approved by the Ethic and Scientific Committee of the Alma Mater Studiorum of the University of Bologna (protocol n. 04/55/09) and subsequently submitted to the Ministry of Health. The study was carried out in accordance with European legislation regarding the protection of animals used for experimental and other scientific purposes (Council Directive 86/609/EEC).
Twelve saddle horses were used in the study. The horses employed were RAO-susceptible horses previously (a year before) employed for similar trials, which developed cough, dyspnoea, pulmonary neutrophilia (> 15% BALF neutrophils) and increased pleural pressure excursions (ΔPplmax > 15 cm H2O – Venti-Graph PG 100/REC - Boehringer Ingelheim) when stabled and exposed to hay indicative of RAO (Ainsworth et al., 2003), without alteration of other parameters of blood count and serum biochemistry parameters over the reference values. During the 6 months prior to the study, the animals were kept in a pasture, vaccinated and dewormed, and showed no clinical signs of RAO. The day before the test, all horses were subjected to pulmonary pressure measurement using the Venti-Graph PG 100/REC (Boeringer Inghelheim), with maximum values of ΔPplmax less than 4 cm H2O.
The horses were randomly divided in two groups: one was left in the pasture and used as the control group (Group B, no. = 4, two males and two females aged 16 to 24 years, mean±SD 22.5±4.4) and the other was the challenged group (Group A, no. = 8, four males and four females aged 17 to 26 years, mean±SD 22.5±3.). With the aim of eliciting the clinical form of RAO, the animals in Group A were moved from the pasture to a poorly ventilated stable with the doors and air vents closed, on unchanged straw, eating hay and, once a day, moldy hay was shaken in front of the horses’ heads in order to increase the environmental powder (re-acutization method based on the previous study of Gigučre et al, 2002, Ainsworth et al 2002 and Ainsworth and Cheetam, 2010).
A clinical examination was performed once a day in each group and a bronchoalveolar lavage was executed in the morning (starting at nine o’clock alternatively among the two groups), before the challenge (T0) to get baseline values, and after 3 (T3), 5 (T5), 8 (T8), 10 (T10) and 12 (T12) days from the onset of the trial. After the bronchoalveolar lavage at T8 the horses of the challenge group were treated with dexamethasone (0.05 mg/kg/iv) once a day for 2 days, and the indoor environment was modified by changing the straw and opening the windows in order to reduce the environmental powder. Each bronchoalveolar lavage was performed after tranquilization with an IV injection of Acepromazine (Prequillan® - 0.2 ml/100 kg) and Detomidine (Domosedan® - 0.1 ml/100 kg) using a BAL catheter (Kruuse, Worldwide Veterinary Supplier - Denmark) passed nasally into the distal respiratory tract. A 210 ml aliquot of sterile pre-warmed (37-38°C) isotonic saline solution, followed by 30 ml of air, was infused and re-aspirated through a 60-mL syringe and then passed into a glass which was kept on ice (Mazan et al., 2003). Each sample was processed within 15 min of collection. The fluid was filtered by means of sterile gauze, centrifuged at 4°C at 2000 x g for 10 minutes and the cell pellets washed twice in phosphate-buffered saline. The sample was then separated into 3 aliquots for cytology, Western blot and RT-PCR, the latter two frozen in nitrogenous up to the time of protein or RNA extraction.
The microscopic evaluation of the BALF cellularity to achieve the differential cells count was determined on a cytospin slide (Cytospin 3, Shandon). Briefly 150 μl of the washed sample were placed in cytochambers and centrifuged at 100 x g for 10 min. The slides were air-dried before staining with May-Grünwald Giemsa quick stain (Diff Quick®, Bioptica, Italy) and a cover slip was placed over the cells using DPX mounting medium. For the differential cell count (DCC), 400 cells were classified under high magnification as macrophages, lymphocytes, neutrophils, eosinophils or epithelial cells, and were expressed as a percentage of the total count.
Western Blot analysis
Frozen BALF samples were lysed in deionised water. Total protein concentration was measured according to the Lowry method using a DC Protein Assay kit (Bio-Rad, Hercules, CA, U.S.A.). Bovine serum albumin (BSA) was used as a standard. Each sample was tested at 750 nm in triplicate using 96-well microtiter plates in a MultisKan EX spectrophotometer.
SDS-PAGE was performed in an Invitrogen Xcell SureLock Mini-Cell using 4-12% Bis-Tris minigels with MES running buffer at pH 7.3, under reducing conditions. Appropriate volumes of the samples were loaded into each well in order to obtain 25 g protein/lane. Each gel was also loaded with standard proteins of known molecular weight (SeeBlue® Plus2, Invitrogen Ltd, Paisley, UK). The electrophoresis system was connected to a power supply (Power Pack Basic – Bio-Rad, Hercules, CA U.S.A.) with a constant voltage of 200 V for 50 min. After electrophoresis, the proteins were transferred to PVDF membranes for 1 h in an Invitrogen Xcell SureLock Blot Module using transfer buffer having a pH of 7.2.
The blots were blocked with concentrated casein solution in buffered saline solution and then incubated with diluted primary specific antibody for 90 min: monoclonal anti-recombinant human IL-1β (2 μg/ml), monoclonal anti-recombinant human IL-8 (2 μg/ml) and monoclonal anti-synthetic β-actin (0,1 μg/ml) (all from Sigma-Aldrich Co). All the primary antibodies were of the IgG1 isotype. The membranes were washed with a specific antibody wash solution (concentrated buffered saline solution containing detergent, from Invitrogen) and incubated with the secondary antibody solution consisting of alkaline phosphatase-conjugated anti-mouse IgG for 45 min. The blots were then visualized using a chromogenic substrate containing BCIP (5-bromo-4-chloro-3-indolyl-1-phosphate) and NBT (nitro blue tetrazolium). Negative controls were obtained by substituting the primary antibodies with an unrelated monoclonal antibody (DAKO Cymation, X0931) of the same isotype (IgG1).
The relative protein content was determined by the ratio between the optic densidy (OD) of the interleukin band and the OD of the reference gene protein using Scion Image software (Scion Corporation, Frederick, Maryland, U.S.A.). Briefly in each interleukin band an area uniform as for the gray level of the pixel was selected and the OD was calculated; the same area was applied to the β-actin band present in the same lane, even in this case aiming to select the field in which the area was uniform and then the OD was obtained. The protein (interleukin) level is expressed in arbitrary units (AU) i.e. the ratio between interleukin OD and the lane matched β-actin OD.
Immunohistochemistry for IL-1β and IL-8
From a horse hospitalized in the Internal Medicine Section of the Department of Veterinary Medical Science with an anamnesis and a confirmed diagnosis of RAO (occurrence of cough and bronchospasm once housed indoors, neutrophils > 15% of the BALF cellularity and pleural pressure excursion > 15 cm H2O) cytospin slides prepared as above were fixed in cold aceton and stored at -80°C until used. For immunohistochemical staining, the slides were hydrated and endogenous peroxidase was blocked by immersion in 0.3% hydrogen peroxide for 20 min. The sections were then rinsed in Tris Buffer and preincubated with a serum-free protein block (Dako, Amsterdam, The Netherlands) for 10 min at room temperature. The primary antibodies (same source as above), anti-human IL-1β diluted 1:20 (in PBS and 0.1% Triton X-100 from Sigma-Aldrich) and anti-recombinant human IL-8 diluted 1:20 (in PBS and 0.1% Triton X-100), were applied for 2 hours at room temperature and were followed by a commercial streptoavidin–biotin-peroxidase procedure (LSAB Kit, Dako, Amsterdam, The Netherlands). Diaminobenzidine (0.05% for 10 min at room temperature) was used as a chromogen. The slides were then stained with May-Grünwald Giemsa quick stain (Diff Quick®, Bioptica, Italy). Negative controls were obtained by substituting the primary antibody with an unrelated monoclonal antibody IgG1 isotype (DAKO Cytomation, X0931) of the same isotype.
RNA extraction and cDNA synthesis
Total cellular RNA was extracted by Trireagent (Sigma, Deisenhofen, Germany) according to the manufacturer’s protocol. All RNA samples were treated with DNAse I (Gibco BRL) in order to remove any trace of genomic DNA contamination. Briefly, 1 U of DNAse I was mixed with 9 μl of total extracted RNA, incubated for 30 min at 37şC and then inactivated for 5 minutes at 95şC. cDNA strands were generated mixing 5 μl of RNA into a reaction buffer with MgCl2 5mM, 10x RNA PCR Buffer 1X, dNTP mixture 1 mM, 0.25 units/μl of Reverse Transcriptase AMV (Takara, Japan), 1 unit/μl of RNase Inhibitor, Random 9 mers 2.5 μM in a final volume of 20 μl of reaction. The retro transcription time was 10’ at 30°C, 15’ at 45°C and 2’ at 99°C.
Quantitative real-time PCR analysis
The Rotor Gene 3000 (Corbett Research, Australia) was used for the amplification and data collection. Primers (Table 1) for gene cytokines (β-actin, IL-1 β, IL-8) were designed by using Oligo software (Molecular Biology Insights, Cascade, CO, U.S.A.). The beta actin gene was used as an internal reference gene to normalize the variation of cell numbers in the bronchoalveolar lavage fluid. The β-actin primers were designed based on the partial equine mRNA sequence. Messenger RNA quantification for each target gene was achieved by constructing standard curves using cDNA of known concentrations of plasmid in which we inserted the target gene through a cloning reaction. The standard curves consisted of serial dilutions of the PCR amplicon corresponding to the gene of interest. Ten-fold dilutions of recombinant plasmid from 106 copies down to 101 copies were used as standards. All concentrations of target gene cDNA were calculated relative to their respective standard curves.
Table 1. Primer sequences used in RT-PCR assays.
Forward primers (5’-3’) Reverse primer (5’-3’) Frame (bp)
IL-1β GAGGCAGCCATGGCAGCAGTA TGTGAGCAGGGAACGGGTATCTT 257
IL-8 TCTCTTGGCCGTCTTCCTG CCGTTGACGAGCTTTACAA 195
Β-actin CTGGCACCACACCTTCTACAACGAG TCACCGGAGTCCATCACGA 214
A real time PCR assay using the Syber Green method was carried out at a final volume of 25 l containing 1X of SYBR PREMIX Ex Taq Takara, 200 nM each forward and reverse primers, 1X of Rox Reference Dye and 2 μl of cDNA. The following were the cycling parameters: 10 min at 95°C for polymerase activation followed by 40 cycles of 15 sec at 95°C, 15 sec annealing (IL-1β 55°C; IL-8 55°C; actin 56°C), 20 sec at 72°C. The signal was acquired on the FAM channel (multichannel machine) (source, 470 nm; detector, 510 nm; gain set to 5) with the fluorescence reading taken at the end of each 72°C step.
To distinguish specific from non-specific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 72 °C for 12 s followed by a gradual increase in temperature to 95 °C at a rate of 0.1°C/s, with the fluorescence signal acquisition mode set to step. All the data are expressed as the ratio (R) between the copies of the target (t) gene and the copies of reference (r) gene actin (Rt-r) [Bowles et al., 2002].
Due to the low number of horses in each group and the data not even close to equal interval scales two nonparametric test have been employed for statistics. The comparison between each point measure (from T3 to T12) and the baseline values (before challenge, at T0) in the challenged group was conducted with the Wilcoxon Matched Pairs Test; the comparison of each point measure between control and challenged groups was done with the Kruskall-Wallis ANOVA median test. Variables compared as above reported were interleukin mRNA Rt-r and AU. Variations in BALF cell percentages were compared, cumulatively across all the trial period (from T0 to T12), between controls and challenged animals by the Kruskall-Wallis ANOVA median test. Linear associations (correlation coefficients - R) between variables were explored and assessed using Pearson’s test. Values of P < 0.05 were considered significant.
The horses in Group A showed evident respiratory distress starting the first day after challenge, with clinical signs of nasal flaring, monolateral or bilateral serous or mucous nasal discharge, coughing, abdominal lift, increased vesicular murmur and, in one case, ocular discharge. The manifestations of the disease decreased quickly after environmental modification and therapy with dexamethasone which followed the bronchoalveolar lavage at T8 and, from T10 to T12, there was a return to the clinical condition similar to that recorded at the pre-trial stage . In Group B, none of the control animals showed any clinical sign of RAO or any clinical manifestation referring to other respiratory disorders during the entire observation period.
In Table 2, the data of the DCC at the different days of sampling of Group A (percentages expressed as the mean±standard deviation calculated among the 8 horses per sampling time) and of Group B (values expressed as the mean±standard deviation of the 4 animals throughout the entire experimental period) are reported. In Group A, at T3, the percentage of neutrophils had increased 15%, in agreement with the previous diagnosis of RAO, and increased constantly thereafter. Other changes included the reduction of lymphocytes and macrophages and the increase of epithelial cells and, later, of eosinophils. In Group B, the DCC maintained values similar to those of the T0 of Group A throughout the entire experiment. Kruskal-Wallis ANOVA median test revealed a significant difference in neutrophils, lymphocytes and epithelial cells when comparing DCC challenged and controls throughout the entire experimental period: the percentage of neutrophils and epithelial cells was higher in Group A (P<0.05) while the percentage of lymphocytes was lower in Group A (P<0.05); other comparisons did not provide any significant results.
Table 2. DCC of the BALF in Group A (values expressed as the mean of the percentages calculated in 8 animals per sampling time) and Group B (values expressed as the mean percentage of 4 animals throughout the entire experimental period).
Time of sampling Neutrophils* Lymphocytes* Macrophages Epithelial cells* Eosinophils
Group A-T0 12.25±16.65 52.50±17.66 29.88±14.46 4.75±4.62 0.63±0.92
Group A-T3 25.50±6.82 29.00±8.32 34.63±10.07 10.50±4.11 0.38±0.52
Group A-T5 27.00±11.88 34.75±16.14 23.50±8.60 14.50±8.59 0.25±0.46
Group A-T8 28.00±14.67 31.00±7.89 25.25±11.16 15.25±8.35 0.50±1.41
Desensitization: dexamethasone/modification of the indoor environment
Group A-T10 30.13±7.97 42.00±16.88 14.38±5.24 11.88±9.13 1.63±2.20
Group A-T12 28.50±21.25 42.13±16.99 16.63±8.02 11.75±9.97 1.00±1.07
Group B - T0/T12 11.75±1.57 53.02±5.81 30.25±3.51 4.22±4.18 0.76±0.89
*P <0.05, Kruskal-Wallis ANOVA median test between challenged and control horses across all the trial (from T0 to T12).
IL-1β and IL-8 mRNA
The lengths of specific fragments of cDNA were 257 bp for IL-1β, 195 bp for IL 8 and 214 bp for β-actin, as expected. In Figures 1 A and B, graphics on the variations of the Rt-r of the mRNA in the challenged animals (Group A) across the trial are shown for IL-1β and IL-8, respectively. As for IL-1β, after values similar to baseline at T3, an increase at T5 is recorded followed by a peak at T8 and a return to baseline values after T10. By the Wilcoxon matched pairs test the IL-1β Rt-r mean value only at T8 resulted significantly higher (P<0.05) than the respective baseline (T0) values. As for IL-8, a surprising peak is present at T0, followed by very low values from T3 to T8; a second peak is present at T10 followed by a low level at T12. By the Wilcoxon Matched Pairs Test none IL8 Rt-r mean value from T3 to T12 resulted significantly higher than the baseline (T0) value. In the control group, the level of cytokine expression showed no significant variation during the study and their Rt-h ranged from 0.0028-0.096 and 0.0095-1.63 for IL-8 and IL-1β, respectively. Significant lower values (P<0.05) of Rt-r in controls compared to challenged horses were revealed at sampling times T0, T3, T5, and T12 for IL-1 β and at sampling times T5, T8, T10 and T12 for IL-8 using Kruskal-Wallis ANOVA median test.
Figure 1. The relationship between IL-1β (A) and IL-8 (B) mRNAs and their respective proteins expressed as RT-PCR Rt-r and western blot AU. Red arrows indicate desensitization.
IL-1β and IL-8 protein assessment
Western blot. Anti-IL-1β antibody revealed a band of approximately 17 kDa, similar to the expected size of equine IL-1β [Howard et al., 1998] (Figure 2a) while anti-IL-8 antibody demonstrated a band of approximately 8 kDa, similar to the expected molecular weight of several specie-specific IL-8 proteins [Lisaza, 2001] (Figure 2b). Reference gene (beta-actin) was revealed by the specific antibody as a band of 42 kDa.
In Figures 1 A and B for IL-1β and IL-8, respectively, graphics on the variations of the levels of the cytokines across the trial (expressed as AU) in the challenged horses are given. As for IL-1β, an increase was registered from the first post-challenge sampling (T3) and was present until the end of the experiment (T12). By the Wilcoxon matched pairs test the IL-1β AU mean values from T3 to T12 resulted significantly higher (P<0.05) than the respective baseline (T0) values. As for IL-8, after values similar to baseline at T3 and T5 a peak was registered at T8, followed by a second peak at T12. By the Wilcoxon Matched Pairs Test only the IL-8 AU T8 mean value resulted significantly higher (P<0.05) than the baseline (T0) value.
In the control group, the cytokine level showed no significant variations during the study, and their AU ranged from 0.00-0.003 to 0.017-0.025 for IL-8 and IL-1β, respectively. Using Kruskal-Wallis ANOVA median test a significant difference (P<0.05) in AU was revealed from T3 to T10 for IL-1β and from T0 to T12 for IL-8 when comparing per sampling times challenged horses and controls, the latter showing values lower than the former.
Figure 2. Western blot analysis of the BALF in 5 horses of the challenged group. A) IL-1β at T3 (lanes 1-5) and T5 (lanes 6-10). B) IL-8 at T5 (lanes 1-5), and T8 (lanes 6-10). Monoclonal antibodies against IL-1β, IL-8 and reference gene (Beta actin) were used. The expected products of approximately 17 kDa (Il-1β), 8 kDa (IL-8) and 42 kDa (beta actin) are indicated close to line 11 (marker).
Immunohistochemistry. IL-1β immunohistochemical stain was observed as a brown cytoplasmic colour of macrophages, epithelial cells and granulocytes (Figures 3 a, b, c), and IL-8 positive stain was detected in the cytoplasm of the epithelial cells and macrophages (Figures 3 e, f).
Figure 3. BALF cytospin. Immunohistochemical staining for IL-1β (a, b, c) and IL-8 (e, f, g). Brown cytoplasmic staining for IL-1β in neutrophils (a), in a macrophage and a neutrophil (b) and in an epithelial cell (c). Negative staining for IL-8 in granulocytes (d), positive staining in macrophages (e) and in 2 out of the 5 epithelial cells in the field (f).
mRNA-protein-BALF cellularity comparisons (Table 3)
In Group A, using the Pearson correlation test, no association between mRNA and protein was found for both IL-1β and IL-8 as well as between the RT-PCR Rt-r of both cytokine mRNA and BALF cell modifications. IL-1β AU were significantly associated with neutrophils and epithelial cells variations (Figures 4 a, b), but not with other BALF cell modifications. Even if no association was appreciable between IL-8 levels of the protein and BALF cell modifications, the peak of IL-8 AU was contextual (T8) with the peak of the epithelial cells in the BALF; then its decrease paralleled the IL-8 protein reduction (Figure 4 c). In Group B, no association was found between the Rt-r and the AU of both cytokines or between the latter and BALF cellularity.
Table 3. Pearson correlation between BALF cell type variations and RT-PRC or WB results in group A horses.
WB IL-1β AU WB IL-8 AU RT-PCR IL8 Rt-r RT-PCR IL-1β Rt-r
Epithelial cells R=0.8399
Figure 4. The relationship between IL-1β western blot AU and neutrophils (a) or epithelial cells (b), or between IL-8 western blot AU and eosinophil (c) variations in the BALF during the experiment. Red arrows indicate desensitization. For statistics, see text.
The triggering of inflammation in RAO is due to 2 main pathways: innate immunity and the late phase of type I hypersensivity. The former mechanism encompasses the ability of alveolar macrophages to acquire a secretive phenotype, particularly capable of secreting several cytokines which initiate and maintain airway inflammation [Franchini et al., 1998]. The late phase of type I hypersensivity plays a central role in a Th mixed response, mainly Th2 at the beginning and Th1 in the late phase of the process [Horohow et al., 2005]. Both ways contribute to triggering inflammation and neutrophils recruitment in the airways by means of several cytokines such as IL-1β and IL-8, respectively [Ainsworth et al., 2006; Laan et al, 2006; Gigučre et al., 2002]. Since the majority of the papers available on heaves concern cytokine gene expression rather than their respective protein assessment, the main aims of this study were to compare mRNA and protein assessments for IL-1β and IL-8 as well as comparing both variables with BALF cellularity variations in horses in which an acute onset of the disease was induced. Then, because a surprising association between IL-1β and neutrophils appeared (Figure 3a), a further sampling from RAO horses, using immunohistochemistry was carried out to demonstrate the presence of these cytokines in the neutrophils.
IL-1β was revealed to have a significant association with neutrophils (Figure 3a) and epithelial cells (Figure 3b) in which a definite immunolocalization was proven. As for the role of equine neutrophils in producing cytokines after being primed with several factors, their ability to produce TNF-α, IL-1β, IL-6, IL-8 and macrophage inflammatory protein 2 (MIP-2) is well-known [Joubert et al., 2001]. Neutrophils in heaves, as well as in other lung pathologies characterized by neutrophil accumulation, acquire the role of modulating inflammation in this way and not only as an effector mechanism recruited in airways. As for the association between IL-1β and epithelial cells, this likely reflects the pro-inflammatory cytokine production during RAO which takes place in the airway epithelium triggered by the NF-Kb transcription factor [Ainsworth et al., 2006]. The cytokines essential for initiating inflammation are considered to be almost exclusively produced by macrophages and a theory has been proposed to explain the exaggerated amounts of pro-inflammatory cytokines produced during RAO episodes by the s.c. “secretory” phenotype of alveolar macrophages [Franchini et al., 1998]. In view of the results here presented, a pro-inflammatory role of the products of both neutrophils and epithelial cells must be taken into consideration. If macrophages play a role in initiating cytokine production, the ability to maintain airway inflammation is instead due to other cell types, namely epithelial cells and neutrophils.
It is well-known that the main producers of IL-8 are the airway epithelial cells [Ainsworth et al., 2006] and a lack of correlation between the above two variables is surprising in our data. However, the peak of IL-8 was recorded when the peak of the epithelial cells was reached in the BALF. Ainsworth et al.  demonstrated a 3-10 fold increase of IL-8 m-RNA in epithelial airway cells and, using immunohistochemistry, its localization in the cytoplasm of epithelial cells in airway biopsies. The amounts of epithelial cells which desquamate and are present in the BALF are not sufficient to demonstrate the role of these cells in the production of IL-8 as appears in the above mentioned study.
The challenge applied to induce RAO was planned according to the criteria proposed by Mc Gorum (1993) and also applied by other Authors [Verfaillie et al., 2001; Kleiber et al., 2005] as well as by our research group in a previous investigation [Pietra et al., 2007], confirming its usefulness for the study of RAO. Both clinical signs and BALF neutrophilia present only in the RAO-susceptible horses of Group A confirmed the induction of the disease by means of the environmental stimulation reproduced in the stalls. The lack of over-expression of cytokines in the RAO-susceptible horses of the control group (Group B) was tied to minimal changes in the respective protein during the entire study and this finding is in agreement with the BALF cytology results. In challenged animals, the trend of mRNA for both cytokines is similar to those of other published studies [Kleiber et al., 2005; Franchini et al., 2000; Gigučre et al., 2002; Pietra et al., 2007] with the peak of IL-1β mRNA followed by IL-8 mRNA. In this investigation, 2 out of the 8 challenged horses had very high levels of IL-8 RT-PCR Rt-r at T0 which raised the mean value of the group as reported in the results. In the literature, high levels of IL-8 m-RNA in RAO-susceptible horses are also reported regardless of treatment with fluticasone as compared to non-treated RAO-susceptible animals [Gigučre et al., 2002]. In the study, similar results are presented concerning the protein (IL-8) which was elevated in RAO-susceptible horses regardless of the sampling time, especially despite normal pulmonary function tests and in the absence of clinical signs of respiratory disease and BAL neutrophilia [Gigučre et al., 2002].
Even if, in our study, the proteins followed the same trend reported for mRNA, this too is also well-known from other studies [Franchini et al., 2000; Gigučre et al., 2002]; surprisingly protein peaks did not follow mRNAs with the unexpected condition to have in some point times evidenced the protein without a previous presence of the related mRNA. This only apparently paradoxical condition can be explained by the lag time in the half-life of these molecules as compared to the sampling. It is well-known that molecular kinetics of cytokines involves hours while the sampling after challenge was planned with intervals of days. For this reason, the time between the samplings has produced some gaps in the demonstration of the mRNA with respect to the protein.
Due to post-transcriptional checkpoints, the presence of mRNA is not conclusive for the production of the transduced product (protein). For this reason, it is important that investigations on cytokines implication in RAO also include protein assessment. In this study, we demonstrated a similarity in the trends of mRNAs and proteins but, to demonstrate a close relationship between the two sets of data, a sampling time closer to the half-life of the cytokines (in hours) would be of benefit.
The increase of IL-1β, paralleled by neutrophil and epithelial cell accumulation in the BALF, is indicative of the active production of the cytokine by these cells. The accumulation of neutrophils is recorded earlier than the increase of IL-8 indicating the presence of different chemokines for neutrophils in the early phase of the disease. Even if the amounts of pro-inflammatory cytokines produced by cells other than macrophages, namely neutrophils and epithelial cells, is low, their elevated number could result in a cumulative amount higher than that of macrophages. This reinforces the pivotal role of cells other than the macrophages in driving airway inflammation during RAO.
The authors are grateful to Gerald Goldsmith for English language editing.
The authors declare that they have no competing interests.
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